Accuprimetmtaq dna polymerase

The V4 hypervariable region of the 16S rRNA genes was amplified using primers 515F and 806R (Apprill et al., 2015 (link); Parada et al., 2016 (link)). PCR was performed using high fidelity AccuPrime^TMTaq DNA Polymerase (Invitrogen, Cat. No. 12346086), including 35 cycles with an annealing temperature of 50°C. PCR clean-up and removal of small fragments was done with the Nucleo Spin Gel and PCR Clean-up Kit (Macherey-Nagel, Cat. No. 740609.250). Quantification of extracted PCR products was performed using PicoGreen assay (QuantIT, Thermo Fisher Scientific, Cat. No. P11496). Thereafter, pooling and sequencing of sample-specific libraries were performed by following the Illumina MiSeq protocol. Data processing was done using the IMNGS platform (Lagkouvardos et al., 2016 (link)), applying the UPARSE analysis pipeline (Edgar, 2013 (link)) with the following settings: Number of allowed mismatches in the barcode: 1, Min fastq quality score for trimming of unpaired reads: 20, Max rate of expected errors in paired sequences: 2, Minimum relative abundance of Operational Taxonomic Units (OTU) cutoff (0-1): 0.25%. The taxonomy of OTUs clustered at 97% sequence identity was determined using SILVA¹ (Pruesse et al., 2012 (link)). The data were submitted to the Sequence Read Archive and are available under the accession number PRJNA514431.

Primer construction for TCRα amplification was based on intestinal transcriptome data, genomic sequences and additional Vα sequence information obtained in the course of the present study (Table 1). Amplification using standard Taq polymerase (Invitrogen) was performed as follows: denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C (30 s), annealing at 55°C (30 s), and extension at 72°C (1 min/1,000 bps), and final extension for 10 min. Amplification using Accuprime^TMTaq DNA polymerase and Accuprime^TM High Fidelity Taq DNA polymerase (Invitrogen) was performed as follows: denaturation at 94°C for 2 min, 30 cycles of denaturation at 94°C (30 s), annealing at 55°C (30 s), and extension at 68°C (1 min/1,000 bps). DNA fragments were excised from the gel and further amplified for 5 cycles before cloning into pCR™ 4-TOPO^® vector (Invitrogen). Sequencing was performed at an in-house sequencing facility using Big Dye termination chemistry (Applied Biosystems).

The use of consented human head and neck cancer tissue samples for this study was approved by the UCLA Institutional Review Board. The HNSCC tissues from two independent patients were obtained from UCLA Department of Pathology & Laboratory Medicine (TPCL). Total RNA was isolated from cells and tissues using TRIzol reagent (Invitrogen), and cDNA was synthesized with random hexamers (Sigma) using SuperScript III reverse transcriptase (Invitrogen). Semi-quantitative RT-PCR was performed using AccuPrimeTM Taq DNA Polymerase according to the manufacturer's protocol (Invitrogen). qRT-PCR was carried out with iQ SYBR green supermix (Bio-Rad) on an iCycler iQ real-time PCR detection system (Bio-Rad). All the primer sequences for PCR are listed (Supplemental Table 1). All samples were run in triplicates in the same culture plate.